The long term objective is to advance the understanding of the role of N-acetylaspartylglutamate (NAAG) in nervous system signaling. Glutamate transmission has been implicated in several types of neurological dsyfunctions, such as excitotoxicity, ALS, schizophrenia, and seizures. How glutamate transmission is regulated is critical to the overall process of synaptic function. NAAG is hypothesized to modulate glutamate transmission at NMDA and mGluR3 receptors. One means of regulating the synaptic action of NAAG is by the neuropeptidase, NAALADase, which hydrolyzes NAAG extracellularly to N-acetyl-aspartate and glutamate. NAALADase has a region of homology to a metalloaminopeptidase, of known structure, from Vibrio proteolytica. Alignment of NAALADase with the Vibrio peptidase suggests specific residues that may coordinate the binding of zinc and substrate. To test the importance of specific residues, substitutions will be made using site-directed mutagenesis. Resultant enzyme kinetics will be quantitated using radiolabeled substrate, and expression of NAALADase will be monitored by immunoblotting in order to normalize enzyme activity. Regulation by phosphorylation will be explored by stimulating cells expressing NAALADase with reagents such as phorbol esters; radiolabeled NAALADase will be immunoprecipitated from a cellular lysate and analyzed by SDS-PAGE. Putative phosphorylation sites in the cytoplasmic domain implicated in enzyme regulation will be targeted for substitution. Regulation of enzyme activity will be explored further using primary rat brain glial cultures; reagents such as phorbol esters will be applied and subsequent NAALADase activity measured.